Active phospholipids as a vector for active molecules

ABSTRACT

An active phospholipid of the formula ##STR1## wherein R 1  is aliphatic of 14 to 24 carbon atoms saturated or containing 1 or 2 unsaturations, R 3  is a residue selected from the group consisting of choline, ethanolamine, glycerol, serine, inositol, ethanol, n-propanol, n-butanol and ethylene glycol and ##STR2## is an active group in the 2-position of the glycerol released by phospholipases and cosmetic and dermatological compositions containing the same useful for treating or caring for the skin.

This application is a division of application Ser. No. 08/364,136 filedDec. 27, 1994 now abn.

STATE OF THE ART

Phospholipids are very widely found in nature as they are the majorconstituents of the walls of animal and vegetable cells. Theirfundamental property is that of being able to form "double layers",constituting the structure of the cells walls. The constitution ofnatural phospholipids is very varied, this variety playing importantroles in cellular metabolisms, roles which are sometimes still unclear.

The structure of phospholipids is the following: ##STR3##

The natural glycerophospholipids contain four ester functions: thecarboxylic ester functions ##STR4## respectively in position 1 and 2 ofthe glycerol on the one hand and between the phosphorus and the variable"polar head", on the other hand, which is constituted by R₃ --O--PO₂--O--. R₁ and R₂ are individually an aliphatic chain of 14 to 24 carbonatoms, saturated or with 1 or 2 unsaturations such as palmitic acid,oleic acid, linoleic acid, stearic acid, or myristic acid and R'3 is aremainder of choline, ethanolamine, glycerol, serine or inositol.

Thus, two fatty acids are esterified at positions 1 and 2 of theglycerol and the 3rd hydroxyl of the glycerol is esterified by aphosphatidyl ester. Position 2 of the glycerophospholipid isparticularly important. In fact, many phospholipase A2's exist intissues, particularly in the skin, so as to release the correspondingfatty acid, ##STR5## which can then be metabolized in the organism.These phospholipase A2's are notably described in Slotta, (1960), TheEnzyme, Vol. 4, p. 552 and Van Deenen et al., (1966), Ann. Rev.Biochem., Vol. 35 (1), p. 674. Furthermore, the exogenous phospholipidshave an affinity for tissues and particularly for the skin, and arecapable of being absorbed into the cell walls.

OBJECTS OF THE INVENTION

It is an object of the invention to provide novel phospholipids offormula I and a process for their preparations.

It is another object of the invention to provide novel cosmetic anddermatological compositions and a novel method of caring for the skin ofwarm-blooded animals including humans.

These and other objects and advantages of the invention will becomeobvious from the following detailed description.

THE INVENTION

The novel phospholipids of the invention have the formula ##STR6##wherein R₁ is aliphatic of 14 to 24 carbon atoms saturated or containing1 or 2 unsaturations, R₃ is a residue selected from the group consistingof choline, ethanolamine, glycerol, serine, inositol, ethanol,n-propanol, n-butanol and ethylene glycol and ##STR7## is an activegroup in the 2-position of the glycerol released by phospholipases.

The cells are provided with functional elements by means ofsemi-synthetic phospholipids: by incorporating functional molecules inposition 2 of a phospholipid. They are rendered particularlybio-available because they will rapidly penetrate the tissues and thenthe cells due to the affinity of their phospholipid residues with thephospholipids of the membranes and will then release the functionalmolecule under the action of the phospholipase A2's.

Examples of an aliphatic of 14 to 24 carbon saturated or containing 1 or2 unsaturations are those derived from oleic acid, palmitic acid,linoleic acid, stearic acid and myristic acid. Examples of R₃ are ethyl,propyl or butyl, preferably ethyl.

The preferred phospholipids of the invention are those of formula Iwherein the active molecule to be grafted at: position 2 is chosen fromthe following molecules: vitamin A acid, all-trans retinoic acid, 9-cisretinoic acid, 13-cis retinoic acid, essential fatty acids such asγ-linolenic acid, α-linolenic acid, eicosapentaenoic acid (EPA),docosahexaenoic acid (DHA), α-hydroxylated acids such as glycolic acid,lactic acid, tartaric acid, α-methyllactic acid, α-hydroxybutyric acid,gluconic acid, mandelic acid, mucic acid, malic acid, α-phenyllacticacid, saccharic acid, tartronic acid; the various acids such as kojicacid, asiatic acid, madecassic acid, benzoic acid, glutamic acid,malonic acid, phytic acid, ascorbic acid, nordihydroguaiaretic acid,salicylic acid, 18β-glycyrrhetinic acid; amino acids such as tyrosine,hydroxyproline, lysine, arginine, the small functional peptides such aspyroGlu-Glu-Asp-Ser-GlyOH or Gly-His-Lys or Arg-Gly-Asp-Ser, or diacidmonoesters such as farnesil succinate, retinol succinate and diacidmonoamides of the formula

    HO.sub.2 --C--(CH.sub.2).sub.n --CO--X--R

in which n is an integer of 2 to 16, X is sulfur, nitrogen or oxygen andR is included in the above list of acids.

A more particular subject of the present invention is activephospholipids as defined above wherein the active molecule to be graftedat position 2 is chosen from the following molecules: vitamin A acid,γ-linolenic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid(DHA), kojic acid, asiatic acid, madecassic acid, glutamic acid, phyticacid, glycolic acid, lactic acid, ascorbic acid, nordihydroguaiareticacid, amino acids such as tyrosine, small functional peptides such aspyroGlu-Glu-Asp-Ser-GlyOH or Gly-His-Lys or Arg-Gly-Asp-Ser and18β-glycyrrhetinic acid.

The novel process of the invention for the preparation of phospholipidsof formula I comprises subjecting a phospholipid or mixtures thereoffrom natural sources of the formula ##STR8## which R₁ and R₂ areindividually aliphatic of 14 to 24 carbon atoms saturated or having 1 or2 unsaturations and R₃ is the remainder of a member of the groupconsisting of choline, ethanolamine, glycerol, serine and inositol to asubstitution reaction to replace R'₃ with a member of the groupconsisting of ethyl, propyl and butyl by enzymatic transphosphatidylation to obtain a product of the formula ##STR9## wherein R₁and R₂ have the above definitions and Z is selected from the groupconsisting of ethyl, propyl and butyl, subjecting the latter toenzymatic hydrolysis of the 2-ester group to obtain a compound of theformula ##STR10## wherein R₁ and Z have the above definition andreacting the latter with an acid anhydride of the formula ##STR11## or acorresponding mixed anhydride in which ##STR12## is a remainder of anactive molecule as defined above, to obtain a compound of formula I.

The starting materials are natural phospholipids, particularly thoseextracted from egg yolk using commercially available or industriallyprepared egg yolk or from soya or soya lecithin.

Generally, the chemistry of the glycerophospholipids is a chemistrycarried out in mixtures of solvents to accommodate the amphipatic natureof these molecules. The products are obtained very dilute and therefore,the costs are high. As a result, practically no use of these products asperfectly defined molecules exists. The preparation process for theactive phospholipids, consists, starting with natural phospholipids, ofacting successively on two of the ester bonds of theglycerophospholipids by replacing the polar head R₃ --O-- and then byselective enzymatic hydrolysis of position 2.

The product of formula I wherein Z is ethyl, propyl or butyl can beoptionally converted by known means to compounds of formula I wherein Zhas the definition of R₃.

In a preferred mode of the process, the natural sources of phospholipidare of animal or vegetable origin such as egg yolk and vegetablelecithin such as soya, preferably phosphatidylcholine orphosphatidylethanolamine or mixtures thereof.

Phosphatidylcholine and phosphatidylethanolamine correspond to a productof formula II in which --O--R'₃ is a remainder of choline ##STR13## anda remainder of ethanolamine ##STR14##

Preferably, the substitution of R'₃ with ethyl or propyl or butyl iseffected by enzymatic transphosphatidylation using phospholipase D inethanol, propanol or butanol [Enzyme Handbook, Barman, p. 545) whichreaction is complete and univocal. The enzymatic hydrolysis to obtainthe ethyl, propyl or butyl lysophosphatidyl of formula IV is effectedwith unpurified phospholipase A₂ in a Ca⁺⁺ ion calcium medium.

The acylation of the hydroxy of formula IV with a biologically activemolecule or molecules to be transported into the membranes is effectedby acidification of the ethanol, propanol or butanol lysophosphatidylfollowed by reaction with an acid anhydride or mixed acid anhydride inan organic solvent such as diethyl ether or toluene.

The originality of this synthesis resides in the close implicationbetween traditional chemical stages and the use of the specificity ofseveral lipolytic enzymes. The principal innovation resides in the useof the intermediate ethanol, propanol or butanol phospholipid (PZ) whichintervenes to facilitate all the stages of the synthesis. The goodenzymatic activities of the phospholipase A2 and of the pancreaticlipase on the phospholipids of this type allow the use of crude enzymesoriginating from the same source: pancreatic powder.

The ethanol, propanol or butanol phospholipid is obtained in a univocalmanner and the method of purification by precipitation which is usedallows a pure product in the form of a calcium salt to be obtained. Theinterest of this protocol relative to those described (Eibl et al.,1981, and Cestaro, et al., 1989) is the obtaining of a calcium salt fromthe (PZ) which can be easily isolated to the exclusion of the sodiumsalt which is formed in the presence of sodium acetate, and the absenceof parasitic products such as phosphatidic acid or non-convertedstarting products. Furthermore, the use of the calcium salt in thefollowing stage which makes use of a hydrolysis catalyzed by strictlycalcium-dependent phospholipase A2 (PA₂) is far more favorable than theuse of phosphatidylethanol in acid form.

A more particular subject of the present invention is a process foractive phospholipids as defined above, characterized in that the activesubstance to be incorporated in the phospholipid in position 2 isselected from the group consisting of vitamin A acid, all-trans retinoicacid, 9-cis retinoic acid, 13-cis retinoic acid, essential fatty acidssuch as γ-linolenic acid, α-linolenic acid, eicosapentaenoic acid (EPA),docosahexaenoic acid (DHA), α-hydroxylated acids such as glycolic acid,lactic acid, tartaric acid, α-methyllactic acid, α-hydroxybutyric acid,gluconic acid, mandelic acid, mucic acid, malic acid, α-phenyllacticacid, saccharic acid, tartronic acid; the various acids such as kojicacid, asiatic acid, madecassic acid, benzoic acid, glutamic acid,malonic acid, phytic acid, ascorbic acid, nordihydroguaiaretic acid,salicylic acid, 18β-glycyrrhetinic acid; amino acids such as tyrosine,hydroxyproline, lysine, arginine, small functional peptides such aspyroGlu-Glu-Asp-Ser-GlyOH or Gly-His--Lys or Arg-Gly-Asp-Ser or alsodiacid monoesters such as farnesil succinate, retinol succinate anddiacid monoamides of general formula

    HO.sub.2 --C--(CH.sub.2).sub.n --X--R

in which n is an integer between 2 and 16, X is sulfur, nitrogen oroxygen atom and R is a group included in the above list of acids, andquite particularly from the following substances: vitamin A acid,γ-linolenic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid(DHA), kojic acid, asiatic acid, madecassic acid, glutamic acid, phyticacid, glycolic acid, lactic acid, ascorbic acid, nordihydroguaiareticacid, amino acids such as tyrosine, small functional peptides such aspyroGlu-Glu-Asp-Ser-GlyOH or Gly-His-Lys or Arg-Gly-Asp-Ser and18β-glycyrrhetinic acid.

Among the products of formula I are the following active phospholipidswhich can be vectors for the active substances as defined above intendedfor particular uses. An active phospholipid as a vector for a retinolester such as vitamin A acid or one of its isomers such as all-transretinoic acid, 9-cis retinoic acid or 13-cis retinoic acid, can be usedto obtain an anti-acne activity, particularly an activity on comedonesor an anti-wrinkle activity.

An active phospholipid as a vector for y-linolenic acid is used for therestoration of the fluidity of membrane walls and for the improvement ofthe condition of the skin of atopic people for whom a lack ofy-linolenic acid causes significant disturbances at the level of theskin. An active phospholipid as a vector for eicosapentaenoic acid (EPA)and/or docosahexenoic acid (DHA) is used for the treatment of psoriasisbecause it has been shown that an exogenous supply of fatty acidsgreatly improves the condition of skin suffering from psoriasis.

An active phospholipid as a vector for an a-hydroxy acid such asglycolic acid, lactic acid, tartaric acid, a-methyl lactic acid,gluconic acid, mandelic acid, mucic acid or malic acid is used for thetreatment of hyper-keratoses, acne, aging, particularly actinic aging.The asiatic and madecassic acids, extracted from the Centella Asiaticaplant are known to have healing and anti-cellulite properties. Theycause the fibroblasts to produce collagen of excellent quality. Graftedand transported by an active phospholipid, their activity is greatlyincreased.

Phytic acid is an excellent chelating agent of metals. Therefore, itpossesses anti-oxidizing, anti-radical and anti-metallo-enzymeproperties, particularly anti-protease properties. Phytic acid can beincorporated in position 2 of an active phospholipid and thus make itmore bio-available. Ascorbic acid or vitamin C is also an excellentanti-radical and anti-oxidizing agent. It participates in all sorts ofbiochemical cycles, particularly in the skin. Incorporated with anactive phospholipid, it is far more stable and more bio-available, veryuseful for example in the case of exposure to solar radiation, where ittraps biologic iron and limits the harmful effects of inflammation.

Nordihydroguaiaretic acid is also an excellent anti-oxidizing agent andit is used for the treatment of sun keratoses. Grafted in position 2 ofan active phospholipid, its bioavailability is considerably increasedwhich allows lower doses to be used for the same activity and thereforethe harmful side effects to be reduced. An active phospholipid carryingtyrosine in position 2 is easily absorbed by the skin and allows adeeper suntan to be obtained because the synthesis of melanine is basedon the use of tyrosine. 18β-glycyrrhetinic acid is a very goodanti-inflammatory but not very soluble and it has difficulty inpenetrating into the skin. Grafted in position 2 of an activephospholipid, its penetration into the skin is greatly enhanced and sois its effectiveness.

The novel cosmetic or dermatological compositions of the invention forthe treatment and care of skin of warm-blooded animals, including humansare comprised of an effective amount of a phospholipid of formula I anda skin care excipient. The compositions are useful for the treatment ofskin suffering from acne, dehydration, wrinkles and damaged skin. Thecompositions preferably contain 0.1 to 10%, more preferably 0.4 to 5%,by weight of the total composition of phospholipid.

Suitable excipients are used to supply these various activephospholipids to the skin. Among the excipients, surfactants arefrequently used such as sorbitan esters like sorbitan stearate,oxy-ethylenated sorbitan esters like POE sorbitan palmitate, sucroseesters like sucrose cocoate, glucose esters or methylglucose estersoxyethylenated or not, like methyl-gluceth-20 or methylglucosesesquistearate, neutralized acyl phosphates like potassiumcetylphosphate, ethoxylated fatty acids like ethoxylated stearic acid,ethoxylated fatty alcohols like ethoxylated stearylic alcohol, more orless de-oiled lecithins from egg or soya, hydrogenated or not andethoxylated vegetable sterols.

The excipients can also contain wetting-agents, preservatives such asmethylparaben, biosol, bronopol, perfumes, coloring agents, fillers suchas talc or polymethacrylate. Optionally, the compositions according tothe invention can also contain insoluble or soluble additives such asliposoluble or hydrosoluble complementary active ingredients, forexample sun filters or solar radiation screens, to give them aprotective power against solar radiation, vitamin extracts andanti-oxidizing agents and dispersing agents and stabilizers.

When the additives are insoluble in the oily and aqueous phases, theytherefore constitute a supplementary phase. They are chosen for examplefrom the following products: perfluoroethers such as FOMBLIN (R) fromthe MONTECATINI Company, insoluble pigments such as: titanium oxides,rutile titanium oxide, anatase titanium oxide, pyrogenated titaniumoxide such as P 25 (R) from Degussa, micronized titanium oxide such asSUN VEIL (R) from Ikeda, titanium oxide treated superficially withsilicones or with amino acids, or with lecithin, or with metallicstearates, iron oxide, iron oxide treated superficially with silicones,or with amino acids, or with lecithin, or with metallic stearates, zincoxide, micronized zinc oxide such as UFZO (R) from Cosmo TrendsCorporation and mica covered with titanium oxide.

Also the dermatological or cosmetic compositions may contain one or morecomplementary liposoluble active ingredients incorporated in the oilyphase of the emulsion, chosen in particular from the followingsubstances for which the preferred percentages are indicated below,expressed relative to the final complete formula:

Vitamin A palmitate: 500 to 10,000 IU/g.

Liposoluble sun filters: octyl methoxycinnamate:

0.5 to 10%, isoamyl ethoxycinnamate: 0.5 to 10%, octyl dimethyl 25 paba:0.5 to 8%, octyl salicylate: 0.5 to 5%, butyl methoxydibenzoyl methane:0.5 to 5%, benzophenone 3: 0.5 to 10%, octyl triazone: 0.5 to 5%, ethyl4-polyethoxy aminobenzoate: 0.5 to 10%, isopropyl 4-dibenzoyl methane:0.5 to 5%.

Nonsaponifiable matter from corn, karite, soya or avocado:

0.1 to 3%.

Ximenoil (R) (oily mixture containing 50% Ximenic acid):

0.1 to 5%, essential extract of sesame oil: 0.1 to 4%, peroxidized cornoil: 0.1 to 10%, tocopherol acetates: 0.05 to 7%, natural tocopherols:0.05 to 5%, farnesol: 0.05 to 5%, linoleic acid 2 to 10%.

Also the dermatological or cosmetic compositions may contain one or morehydrosoluble complementary active ingredients incorporated in theaqueous phase of the emulsion, chosen notably from sodium lactate,extracts of Hafnia biolyzate, extracts of Klebsellia pneumoniaebiolyzate and hydrosoluble sun filters.

The hydrosoluble complementary active ingredients can also be chosenfrom the above substances for which the preferred percentages areindicated, expressed relative to the final complete formula:

neutralized 2-phenyl benzimidazol 5-sulfonic acid: 0.5 to 8%

neutralized 2-hydroxy 4-methoxybenzophenone 5-sulfonic acid: 0.5 to 5%

Ascorbic acid: 0.5 to 10%, caffeine benzoate: 0.1 to 5%, phytic acid:0.1 to 5%, mucic acid: 0.1 to 5%, hydrolysates of vegetable proteins:0.1 to 10%, polyglucan: 0.1 to 5%

Mexican mimosa extract: 0.5 to 20%, chitosan: 0.5 to 20%, marine animalserum: 0.1 to 3%

Hirudin extract: 0.5 to 10%, meristem extract: 0.1 to 5%, procyanodolicoligomers: 0.05 to 3%, yeast extracts: 0.05 to 3%, panthenol: 0.05 to5%, centella asiatica extract: 0.05 to 3% glycyrrhetinic acid: 0.05 to2%.

The cosmetic compositions of the invention can be presented in all theforms used in cosmetology: creams or gels in pots or in tubes, milks inglass or plastic bottles and optionally in dosing-bottles or phials.

The dermatological compositions as defined above can be presented in theform of liquid or solid preparations for topical use and particularly inone of the following forms:

fatty gels,

simple, water-in-oil emulsions,

simple, oil-in-water emulsions,

multiple emulsions, for example a triple water-in-oil--in-water oroil-in-water-in-oil emulsion,

complex emulsions containing liquid crystals forming lipidic doublelayers surrounding the oily phases,

an oil-in-water emulsion containing liquid crystals,

oil-in-water or water-in-oil micro-emulsions,

emulsions containing dispersed oily phases, which are different from andinsoluble with each other,

pseudo-emulsions or dispersion of an oily phase in an aqueous phase andstabilized with gelatinizing agents such as Lubragel (R), (polyglycerylmethacrylate marketed by SEDERMA, FRANCE), Pemulen (R), Hypan (R),Xanthan gum, CMC, hydroxyethyl cellulose, Amigel (R),Polyvinylpyrrolidone, Amercell HM1500 (R), or a mixture of two or moreof these gelatinizing agents, without traditional surfactants.

The method of treating comedones comprises applying to comedones aneffective amount of a phospholipid of formula I containing a vector fora retinal ester such as vitamin A acid or one of its isomers such asall-trans retinoic acid, 9-cis retinoic acid.

The method can also be used to treat dry or dehydrated skin or to supplythe skin with living organisms and active substances.

In the following examples, there-are described several preferredembodiments to illustrate the invention. However, it is to be understoodthat the invention is not intended to be limited to the specificembodiments.

EXAMPLE 1 THE PREPARATION OF A PHOSPHOLIPID AS A VECTOR FOR VITAMIN AACID

The conversion of a mixture of phopholipids extracted from a naturalsource into a vector compound for vitamin A acid is carried out by athree-stage process:

1) Extraction of the natural phospholipids and their conversion into asingle compound,

2) Selective hydrolysis in position 2,

3) Reacylation of the lyso-phospholipid.

The following abbreviations will be used:

PC: phosphatidylcholine: ##STR15## PE: phosphatidylethanolamine:##STR16## PET: phosphatidylethanol: ##STR17## in which R₁ and R₂ havethe above definitions.

1) Extraction of the natural phospholipids; and their conversion into asingle compound.

a) Extraction of the natural phospholipids

The phospholipid source used was chicken egg yolk withcommercially-available eggs or industrially-prepared egg yolk beingused. Starting with commercially-available eggs, the first stage wastheir hardening achieved at about 100° C. over about 10 minutes. Theyolks were then collected and extraction was carried out either directlyor after drying. The extraction was carried out first with 3 extractionswith 200 ml of acetone for 400 g of wet yolk by simple stirring of thecrushed yolk in the solvent. The suspension was filtered and the solidwas treated again with acetone. The creamy-white residual solid wasdried and dispersed 3 times in 200 ml of ethanol. After filtration, theethanolic phase was either preserved in the volume necessary for thefollowing stage, or evaporated under reduced pressure. The residue wasdiluted in a minimum of dichloromethane (DCM) and 500 ml of acetone wereadded. The phospholipids precipitated and the suspension was kept at 4°C. for 16 hours, then filtered and dried.

The extraction rate was on average 1 g to phospholipids per egg yolk and150 g per kilo of dry product were obtained. The mixture was constitutedfor the main part by PC and PE in the proportions of 4 to 1.

b) Conversion of the PC/PE mixture into PET ##STR18## in which R₁ and R₂have the above meanings.

This conversion was carried out using the property of phospholipase D(PLD) to catalyze the transphophatidylation reactions which enable thepolar head of the phospholipids to be modified. The PLD was extractedfrom cabbage leaves by chopping 1 kg of white leaves using a "blender"with 1 liter of distilled water. The ground-up product was filtered andthe filtrate of 1.1 to 1.2 liters was used as is or diluted. 40 mM ofcalcium chloride were added and the pH was between 5 and 6.

A half volume of an ethanolic solution of the PC/PE mixture in avariable quantity of 10 to 80 g/l, preferably 40 g/l, was added to onevolume of enzymatic extract with vigorous stirring. The development ofthe reaction was monitored by thin layer chromatography (TLC). After atime varying from 2 to 10 hours, the disappearance of the PC and the PEand the formation of a gummy precipitate were observed. The aqueousphase was eliminated and the precipitate was dissolved in the minimum ofdichloromethane or other appropriate solvent, filtered, then purified byprecipitation in 400 ml of acetone with stirring. Filtration and dryingwere carried out to obtain a creamy white product in a yield of 60 to90%, which was the calcium salt of phosphatidylethanol.

2) Hydrolysis in position 2 of the PET to obtain the lyso-PET ##STR19##in which R₁ and R₂ have the above meanings.

The source of PA2 used was porcine pancreatic powder (Fluka) and 2 g ofenzymatic powder were dissolved in 10 to 20 ml of a solution of 50 mM ofboric acid and 50 mM of calcium chloride. The pH was adjusted to 2 to 3over 20 minutes and then the pH was returned to 7 to 8 with 1N sodiumhydroxide. A solution of 10 g of PET was then added in the form of 16mmol of the calcium salt in 100 to 200 ml of ether and the mixture wasvigorously stirred. The development of the reaction was monitored bythin layer chromatography and a regular disappearance of the PET infavor of the lyso-PET and fatty acids released by the hydrolysis wasobserved. At the end of the reaction, filtration was carried out oncelite. To purify the lyso-PET by precipitation, the ether wasevaporated so that only a sufficient volume for the solubility of thesolution remained and the solution was poured into 400 ml of acetonewith vigorous stirring. The lyso-PET in the form of the calcium salt,creamy white in color, precipitated while the released fatty acids weresoluble in the acetone. Filtration and drying were carried out to obtainthe lyso-PET derivative in a yield of 60 to 90%.

3) Reacylation of position 2.

This stage was divided into two sub-stages: acidification of thelyso-PET and actual acylation after optionally obtaining in situ ananhydride of vitamin A acid.

A) Acidification of the lyso-PET.

The lyso-PET previously obtained contained salt forms and the acid formwas obtained by dissolution of 5 g of the product in 10 ml of chloroformor dichloromethane and 12 ml of methanol mixture and washing 3 timeswith 10 ml of iN hydrochloric acid. The organic phase was washed withdistilled water and evaporated under reduced pressure in the presence oftoluene. The crude product was dried under reduced pressure in adessicator.

b) Actual acylation after optionally obtaining in situ an anhydride ofvitamin A acid.

The acylation of the acid form of the lyso-PET was carried out using theacid that it is desired to introduce in activated form. For high valueacids such as retinoic acid or polyunsaturated acids, a mixed anhydridewith pivalic acid or dicyclohexylcarbodiimide (DCC) in the reactionmedium was prepared. In the case where a mixed anhydride was prepared,it was the acyl of the mixed anhydride which was at least stericallyhindered which transferred onto the receiving alcohol.

Acylation by the anhydride ##STR20## Preparation of the anhydride: 0.2ml (1.6 mmol) of pivaloyl chloride were added to a solution of 500 mg ofvitamin A acid (1.66 mmol) in 20 ml of toluene and ethyl ethercontaining 1 ml of triethylamine (7.2 mmol). The reaction was carriedout shaded from the light and under an inert atmosphere. Monitoring byTLC indicated the disappearance of the acid in a few minutes.

Acylation ##STR21## in which R₁ has the meaning indicated above.

Next, 600 mg (1.3 mmol) of the lyso-PET dissolved in 10 ml of tolueneand ethyl ether and 12 mg of 4-dimethylamino-pyridine (0.13 mmol) and 1ml of toluene or ethyl ether were added. The development of the reactionwas monitored by TLC and the reaction was complete (disappearance of thelyso-PET) after 6 to 10 hours. Filtration was carried out on celite and10 ml of 1N hydrochloric acid were added. The organic phase was washedwith water and evaporation was carried out in the presence of toluene.The crude mixture was fractionated by chromatography on silica and theyield after purification was 60% to obtain 600 mg of PET-vitamin A.

Acylation with dicyclohexylcarbodiimide (DCC) ##STR22##

600 mg (1.3 mmol) of lyso-PET were dissolved in 10 ml of toluene andethyl ether and 12 mg of (0.13 mmol) in 1 ml of toluene or ethyl etherand 322 g (1.56 mmol) of dicyclohexylcarbodiimide were added to asolution of 500 mg (1.66 mmol) of vitamin A acid in 20 ml of toluene orethyl ether containing 1 ml of triethylamine (7.2 mmol). The developmentof the reaction was monitored by TLC and the reaction was complete(disappearance of the lyso-PET) in 6 to 10 hours. Filtration was carriedout on celite and 10 ml of 1N hydrochloric acid were added. The organicphase was washed with water and evaporation was carried out in thepresence of toluene. The crude mixture was fractionated bychromatography on silica to obtain a yield after purification of 60% or600 mg of PET-vitamin A.

EXAMPLE 2

The following oily phase was heated to 70° C:

    ______________________________________                                        Stearamidopropyl PG-dimonium chloride                                                               3.0            g                                        Phosphate (CTFA name)                                                         Cocamidopropyl PG-dimonium chloride                                                                 1.0            g                                        Phosphate (CTFA name)                                                         Cetyl alcohol         3.0            g                                        Myristyl myristate    5.0            g                                        Hydrogenated polyisobutene                                                                          2.0            g                                        Karite butter         2.0            g                                        Propylene glycol stearate                                                                           3.0            g                                        Silicone oil          2.0            g                                        Vegetable oil         5.0            g                                        Anti-oxidant          0.2            g                                        Oleyl acetate         1.0            g                                        ______________________________________                                    

Furthermore, the following aqueous phase was prepared and was heated to70° C.:

    ______________________________________                                        Demineralized water SQF                                                                           100.0          g                                          Glycerin            10.0           g                                          Modified hydroxy ethylcellulose                                                                   0.5            g                                          PVP                 1.0            g                                          Preservatives       0.52           g                                          ______________________________________                                    

The oil-in-water (O/W) emulsion was prepared by vigorously mixing thetwo phases at 90° C. for 10 minutes, followed by slowly cooling undermoderate stirring. 0.5% of perfume, then 0.5% of all-trans 2-RetinoylPhosphatidyl Ethanol were added at 45° C. The mixture was cooled to 25°C. and this cationic O/W emulsion was used in the treatment of acne.

EXAMPLE 3

Cream for the treatment of atopic skins

    ______________________________________                                        γ linolenyl Phosphatidyl Ethanol                                                            5.0            g                                          Potassium alkyl phosphate                                                                         2.0            g                                          Ethyl hexyl palmitate                                                                             8.0            g                                          Hydrogenated lanolin                                                                              5.0            g                                          Triglycerides of fatty acids                                                                      4.0            g                                          Sorbitan stearate   1.0            g                                          Neutralized carboxyvinyl                                                                          0.4            g                                          polymer                                                                       Preservatives       0.4            g                                          Purified water SQF  100.0          g                                          ______________________________________                                    

EXAMPLE 4

Cream for the treatment of psoriasis

    ______________________________________                                        2-EPA Phosphatidyl Ethanol                                                                        1.0            g                                          2-DHA Phosphatidyl Ethanol                                                                        1.0            g                                          Cylcerol stearate   4.0            g                                          Sorbitan palmitate  6.0            g                                          Perhydrosqualene    5.0            g                                          Diisopropyl-cyclohexane                                                                           7.0            g                                          Capric/caprylic triglycerides                                                                     9.0            g                                          Glycerin            5.0            g                                          Preservatives       0.35           g                                          Purified water SQF  0.35           g                                          ______________________________________                                    

EXAMPLE 5

Sun lotion

    ______________________________________                                        2-Ascorbyl Phosphatidyl ethanol                                                                   3.0            g                                          Sun filters         5.0            g                                          Vaseline oil        10.0           g                                          Cetearyl octanoate  4.0            g                                          De-oiled soya phospholipids                                                                       5.0            g                                          Silicone oil        2.5            g                                          Cetyl ether P.O.E.  2.0            g                                          Sorbitan stearate   1.0            g                                          Preservatives       0.35           g                                          Aromatic composition                                                                              0.5            g                                          Purified water SQF  100.0          g                                          ______________________________________                                    

EXAMPLE 6

Multiple emulsion for the treatment of actinic aging.

The following aqueous phase, called the internal aqueous phase, washeated to 80° C.:

    ______________________________________                                        Demineralized water      26.52        g                                       Methylparaben            0.1          g                                       Magnesium sulfate        0.28         g                                       Glycerin 30' B           0.8          g                                       O-cymen-5-ol             0.04         g                                       The following oily phase was heated separately:                               Glyceryl isostearate     2.0          g                                       Polyoxyethylenated hydrogenated ricin                                                                  0.2          g                                       oil (7 mols)                                                                  Soya oil                 8.2          g                                       Propylparaben            0.06         g                                       Volatile silicone oil    1.6          g                                       2-Lactyl Phosphatidyl ethanol                                                                          5.0          g                                       ______________________________________                                    

The aqueous phase was dispersed in the oily phase at 80° C. byvigorously stirring for 5 minutes and then the mixture was cooled slowlyto 25° C.

Then, this primary water/oil emulsion was dispersed in the followingaqueous phase, called the external aqueous phase, by mixing gently atambient temperature:

    ______________________________________                                        Demineralized water SQF                                                                         100.0            g                                          Lubragel MS (R)   15.0             g                                          Carbopol 980 (R)  0.03             g                                          Tetrasodium EDTA  0.054            g                                          Methylparaben     0.216            g                                          Imidazolidinyl urea                                                                             0.216            g                                          Pure sodium hydroxide                                                                           0.125            g                                          ______________________________________                                    

EXAMPLE 7

Twin-phase healing emulsion

The following oily phase was heated to 80° C.:

    ______________________________________                                        Stearyl alcohol          1.0          g                                       Cetyl alcohol            2.0          g                                       Cetearyl octanoate       4.0          g                                       Polysorbate 60           4.0          g                                       Sorbitan stearate        4.0          g                                       Safflower oil            6.0          g                                       Karite butter            3.0          g                                       Silicone oil             0.5          g                                       Tocopherols              0.05         g                                       2-Asiaticosyl Phosphatidyl Ethanol                                                                     0.5          g                                       2-Madecassyl Phosphatidyl Ethanol                                                                      0.5          g                                       The following aqueous phase was heated to 80° C.:                      Demineralized water SQF  100.0        g                                       Carboxyvinyl polymer     0.3          g                                       Preservative             0.7          g                                       Lubragel MS (R)          5.0          g                                       Pure sodium hydroxide    0.3          g                                       ______________________________________                                    

The oily phase was dispersed in the aqueous phase and the mixture wasstirred vigorously for 10 minutes. The emulsion thus formed was thenslowly cooled to 25° C.

EXAMPLE 8

Water/silicone emulsion for the treatment of sun keratoses

The following oily phase was heated to 60° C.:

    ______________________________________                                        Demineralized water SQF  100.0        g                                       Sodium chloride          0.8          g                                       Pure citric acid         0.01         g                                       Methylparaben            0.25         g                                       Propylene glycol         2.0          g                                       O-cymen-5-ol             0.1          g                                       The following silicone phase was heated to 60° C.:                     Isocetyl stearate        3.0          g                                       Arlacel 83 (R)           0.8          g                                       Hydrogenated ricin oil   0.3          g                                       Elfacos ST9 (R)          2.0          g                                       2-NDGA Phosphatidyl Ethanol                                                                            2.0          g                                       DC Silicone 3225 (R) (DOW CORNING)                                                                     9.0          g                                       Volatile silicone        4.0          g                                       ______________________________________                                    

The aqueous phase was dispersed in the silicone phase under moderatestirring for 10 minutes and the emulsion formed was cooled to 25° C.

EXAMPLE 9

Pre-tanning emulsion without emulsifier

The following oily phase is heated to 80° C.:

    ______________________________________                                        Wheatgerm oil            4.0          g                                       Polyisobutene            4.0          g                                       Octyl stearate           4.0          g                                       Ceramide HO3             2.0          g                                       2-Tyrosinyl Phosphatidyl Ethanol                                                                       2.0          g                                       The following aqueous phase was heated to 80° C.:                      Glycerin 30' codex       3.0          g                                       Carboxyvinyl polymer     0.45         g                                       Lubragel MS (R)          4.0          g                                       Pure sodium hydroxide    0.055        g                                       Preservatives            0.55         g                                       Perfume                  0.20         g                                       Demineralized water      60.0         g                                       ______________________________________                                    

The oily phase was dispersed in the aqueous phase with very weakstirring and high shearing for 30 minutes. Then, the emulsion formed wasslowly cooled to 45° C., and the following was added under strongstirring:

    ______________________________________                                                Talc 3.0 g                                                            ______________________________________                                    

When the dispersion of the talc had completely finished, cooling of themixture was continued under slow stirring. When the temperature was 25°C., the following was added under moderate agitation:

    ______________________________________                                                Perfume                                                                             0.2 g                                                           ______________________________________                                    

EXAMPLE 10

Anti-inflammatory emulsion

An oil-in-water emulsion was prepared in the following manner.

The following oily phase was heated to 80° C.:

    ______________________________________                                        self-emulsifying glycerol stearate                                                                     6.0          g                                       (Arlacel 165 (R) from the ICI company)                                        Cetyl alcohol            1.0          g                                       Sterol of ethoxylated soya                                                                             2.0          g                                       (Generol 122 E 10 (R) from the Henkel company)                                Mixture of vaseline oil and lanolin alcohol                                                            3.0          g                                       (Amerchol L101 (R) from the Amerchol company)                                 Petrolatum and lanolin alcohol                                                                         1.0          g                                       (Amerchol CAB (R) from the Amerchol company)                                  Olive oil                6.0          g                                       Karite butter            3.0          g                                       Propyl paraben           0.05         g                                       2-glycyrrhetinyl Phosphatidyl Ethanol                                                                  1.0          g                                       ______________________________________                                    

The following aqueous phase was heated to 80° C.:

    ______________________________________                                        Demineralized water                                                                           60.0              g                                           70% Sorbitol    3.0               g                                           Xanthan gum     0.3               g                                           Methylparaben   0.1               g                                           ______________________________________                                    

When the Xanthan gum was well dispersed, the oily phase was added to theaqueous phase at 80° C., and vigorous stirring was carried out for 20minutes to form the emulsion. Then, the stirring was reduced and theemulsion was slowly cooled to 40° C. Then, 2 g of water containing 0.15g of imidazolidinyl urea and then 0.3 g of perfume were added to it.

TEST OF THE ACTIVITY OF AN ACTIVE PHOSPHOLIPID

As an example, the comedolytic power of all-trans 2-retinoylphosphatidinyl ethanol was tested following the protocol below:

The animal chosen for the test of comedolytic activity was the HairlessRhino (hr rh) mouse of female sex, this choice being due to the factthat the skin of such an animal has a high density of comedones having alarge diameter and narrow orifice. The use of comedolytic agents on theskin of the animal provokes the opening of the orifice of the comedones,the release of the horny material and the sebum that it contains.

Two groups comprising six mice each which were six-week's old at thestart of the test and weighing on average 18 grams each were used. Thefirst group were mice treated with distilled water (negative controlgroup) and the second group were mice treated with the product understudy. The treatment consisted of a topical use of the product studiedon the interscapular area at a dose of 0.02 ml, 5 days out of 7, for 21days. The animals were sacrificed at the end of three weeks oftreatment, 24 hours after the last application.

Biopsies of the skin were then taken from the treated areas of theanimals and from these biopsies, sections were prepared for a standardmorphometric study by methods known to a man skilled in the art. Thefollowing parameters were measured: diameter of the opening of thecomedon at the surface i.e d

    ______________________________________                                        diameter of the comedon                                                                             i.e D                                                   comedonian profile    i.e R = d/D                                             ______________________________________                                    

The ratio R=d/D allowed the action of the comedolytic agents to bequantified.

The percentage of inhibition of the comedones was calculated for theproduct under study relative to the negative control, i.e. the followingratio: ##EQU1##

The comedolytic activity of all-trans 2-retinoyl phosphatidinyl ethanolat 0.1% in solution in ethylenediglycol was measured by this method andproduced the following results:

    ______________________________________                                        Product R =       0.89                                                        Negative control R =                                                                            0.78                                                        % inhibition =    14.1%                                                       ______________________________________                                    

Therefore it can be seen that all-trans 2-retinoyl phosphatidinylethanol at a dose of 0.1% had a good comedolytic activity.

Various modifications of the compositions and method of the inventionmay be made without departing from the spirit or scope thereof and it isto be understood that the invention is intended to be limited only asdefined in the appended claims.

What is claimed is:
 1. A method of treating a skin condition of awarm-blooded animal, comprising applying to the skin a skincondition-treating effective amount of a phospholipid of the formula##STR23## wherein R₁ is an aliphatic residue having 14-24 carbon atomswhich is saturated or contains 1-2 saturations, R₃ is a residue derivedfrom a compound selected from the group consisting of ethanol,n-propanol and n-butanol and Y--C(O)-- is an active group which iscapable of release by phospholipases.
 2. The method of claim 1,whereinY--C(O)-- is an active group derived from an acid selected fromthe group consisting of vitamin A acid, all-trans retinoic acid, 9-cisretinoic acid, 13-cis retinoic acid, γ-linolenic acid, α-linolenic acid,eicosapentaenoic acid, docosahexaenoic acid, glycolic acid, lactic acid,tartaric acid, α-methyllactic acid, α-hydroxybutyric acid, gluconic,acid, mandelic acid, mucic acid, malic acid, α-phenyllactic acid,saccharic acid, tartronic acid, kojic acid, asiatic acid, madecassicacid, benzoic acid, glutamic acid, malonic acid, phytic acid, ascorbicacid, nordihydroguaiaretic acid, salicylic acid, 18β-glycyrrhetinicacid, tyrosine, hydroxyproline, lysine, arginine,pyroGlu-Glu-Asp-Ser-Gly, Gly-His-Lys, Arg-Gly-Asp-Ser, farnesilsuccinate, retinol succinate and diacid derivative of the formula

    HO.sub.2 --C--(CH.sub.2).sub.n --CO--X--R

wherein n is 2-16, X is selected from the group consisting of sulfur,nitrogen and oxygen and R is a group of formula Y--C(O)-- as definedabove.
 3. The method of claim 1, whereinY--C(O)-- is an active groupderived from an acid selected from the group consisting of vitamin Aacid, y-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid,kojic acid, asiatic acid, madecassic acid, glutamic acid, phytic acid,glycolic acid, lactic acid, ascorbic acid, nordihydroguaiaretic acid,18βglycyrrhetinic acid, tyrosine, pyroGlu-Glu-Asp-Ser-Gly, Gly-His-Lysand Arg-Gly-Asp-Ser.
 4. The method of claim 1, wherein the skincondition is selected from the group consisting of acne, comedones,wrinkles, psoriasis, hyperkeratoses, dehydration and sunburn.
 5. Themethod of claim 1, wherein Y--C(O)-- is an active group derived from anacid, wherein the acid is vitamin A acid, or an isomer thereof.
 6. Themethod of claim 1, further comprising co-applying to the skin vitamin Apalmitate.
 7. The method of claim 1, further comprising co-applying tothe skin at least one liposoluble sun filter.
 8. The method of claim 7,wherein the at least one liposoluble sun filter is at least one memberselected from the group consisting of octyl methoxycinnamate, isoamylethoxycinnamate, octyl dimethyl 25 paba, octyl salicylate, butylmethoxy-dibenzoyl methane, benzophenone, octyl triazone, ethyl4-polyethoxy aminobenzoate and isopropyl 4-dibenzoyl methane.
 9. Themethod of claim 1, further comprising co-applying to the skinnonsaponifiable vegetable matter.
 10. The method of claim 9, wherein thevegetable matter is selected from the group consisting of corn, karite,soya and avocado.
 11. The method of claim 9, further comprisingco-applying to the skin at least one additional ingredient selected fromthe group consisting of ximenic acid, tocopherol acetates, naturaltocopherols, farnesol, linoleic acid and sodium lactate.
 12. The methodof claim 1, further comprising co-applying to the skin at least oneadditional ingredient selected from the group consisting of sesame oiland peroxidized corn oil.
 13. The method of claim 1, further comprisingco-applying to the skin at least one additional ingredient selected fromthe group consisting of extract of Hafnia and extract of Klebselliapneumoniae.
 14. The method of claim 1, further comprising co-applying tothe skin at least one hydrosoluble sun filter.